As a medium for chromatography to be used for adsorption, separation and purification of proteins, an inorganic medium represented by a silica compound or the like and an organic medium comprising an organic polymer may be mentioned. The organic medium is roughly classified into a synthetic medium using a synthetic compound represented by a (meth)acrylate and a (meth)acrylamide, and a natural medium using a natural polysaccharide represented by agarose, dextran, mannan or the like.
A synthetic medium is usually produced, for example, by suspension polymerization method using a mixed liquid of a monofunctional monomer with a polyfunctional monomer, such as glycidyl methacrylate with ethylene glycol dimethacrylate, followed by hydrophilization by a water soluble polyhydric alcohol or the like to produce a substrate. By the progress of hydrophilization technology in synthetic media in recent years, a process for producing a synthetic medium comparable to a natural medium in view of hydrophilicity has been found. Further, the process is usually a process for producing particles by suspension polymerization using a monofunctional monomer and a polyfunctional monomer, a hard medium and a flexible medium can be freely designed by adjusting the amount of addition of the polyfunctional monomer. However, with a view to producing a medium which is hard and which is not fragile, in the case of a synthetic medium, it is common to produce a medium by using a (meth)acrylate or (meth)acrylamide monomer with which a polymer having a high molecular weight is easily obtained. Further, with a view to easily attaching a ligand which interacts with proteins after formation into particles, many media using a so-called glycidyl methacrylate (GMA) monomer having a glycidyl group to be the reaction site have been proposed.
As described above, the main purpose of use of such a medium is separation and purification of proteins by liquid chromatography. The main purpose of use of the purified proteins is pharmaceutical preparations (injectable protein preparations), and in this field, it is required to completely avoid risks of side effects by contamination by contaminants.
The contaminants which may be included may, for example, be (1) contaminants derived from the aimed protein production process, such as foreign proteins, nucleic acids, endotoxins and viruses contained in culture solutions, blood sera, etc., (2) contaminants from components necessary for production and storage, such as additives, and from equipment, media, separation membranes, solutions, etc. used in the purification step, and (3) components which have been adsorbed on and not eluted from the medium in the previous purification.
The medium is packed in a column and used in a purification step, and it is necessarily washed with a verified washing method before first use and before reuse. The most common method of washing the interior of an apparatus in the purification step is washing with 1N sodium hydroxide, which can decompose and wash away proteins, endotoxins, etc. This washing method is recommended as a guideline of Food and Drug Administration, and is effective at the first use and at the reuse. That is, in GMP facility to carry out purification of pharmaceutical proteins, it is commonly carried out to wash the interior of the column apparatus before each batch of use. Further, in a case where there is an interval between batches, the apparatus may be filled with an aqueous sodium hydroxide solution diluted to 0.01N to 0.1N to shut off the apparatus in some cases. Further, to remove abnormal proteins such as prion, washing with an aqueous sodium hydroxide solution at a higher concentration (e.g. a concentration of 2N) is required.
However, a GMA monomer has an ester moiety showing high hydrophilicity and accordingly hydrolysis of ester proceeds by a long time contact with an alkaline chemical, whereby an alcohol compound is released to form a carboxylic acid. There will be no problem if the released alcohol compound can be completely removed by washing, but if it can not be removed, it may be a contaminant. Further, by formation of a carboxylic acid, the original properties of the medium may change, and the reproducibility of separation and purification will disappear, thus leading to poor purity. That is, a medium having low alkali resistance not only has a short period of use by deterioration of properties but also has problems such as risks of unknown elutes, risks of contamination by elution at the time of purification operation and poor purity by deterioration of properties.
As described above, although a synthetic medium is hard (has high mechanical strength) and is thereby has such an advantage that it is suitable for high speed/high separation, and has such an advantage of high hydrophilicity, development of a synthetic medium having alkali resistance has been expected at present.
Patent Document 1: JP-B-58-058026
Patent Document 2: JP-A-53-090991
Patent Document 3: JP-A-05-009233